GSM7118635: Lateral choroid plexus, Postnatal day 1, wild type, pool 2; Mus musculus; RNA-Seq
All animal procedures were conducted in accordance with NIH Guidelines and approved by the UF IACUC. Mbnl2-/- knockout (Mbnl2 KO) mice were generated previously and bred to obtain congenic (N10) animals on the FVB/NJ background. Timed mating's were performed to obtain littermate wild-type FVB/NJ and Mbnl2-/- (KO) prenatal timepoint embryos, and embryonic timepoints (E13.5) were collected followed by choroid plexus (ChP) isolation. Postnatal timepoint (P1, P14, and P63) mice were euthanized and the ChP isolated. For ChP isolation, mice were euthanized, and the brain carefully transferred to cold PBS. To isolate the LVChP, the brain was cut coronally approximately 1/3 from the top to open the lateral ventricles. The lateral ventricles were identified and carefully opened to allow visualization and removal of the LVChP by gentle separation along the base. Individual ChP samples were genotyped, sex identified, and RNA isolated. Before library preparation, RNA samples were evaluated and pooled (2-4) for sufficient RNA. All experiments prior to P21 were comprised of equal numbers of males/females, in contrast to P21+ which were all males. Females were needed to generate additional embryonic timepoints. For RNA isolation, tissue samples were homogenized in 300 μL TRI Reagent (Zymo R2050-1-200) and incubated at 25ºC for 10 min. Total RNA was isolated using the Direct-zol RNA miniprep kit (Zymo), following the manufacturer's instructions, including an on-column DNase I digestion step. RNA concentration was determined using the Qubit 4 Fluorometer with a broad range RNA quantification broad range kit (Invitrogen). RNA quality was checked using a Fragment Analyzer (Agilent) and depleted of rRNA (rRNA Depletion Kit v2 Human/Mouse/Rat, NEB). cDNA libraries prepared using the Ultra II Directional RNA Library, NEB. Mouse developmental ChP libraries were generated using 168 ng of total RNA input and sequenced on Illumina NextSeq500 platform.